Bottom Line:
Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake.SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat.These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Affiliation: Department of Genetics, The University of Melbourne, Victoria, Australia.

ABSTRACTDietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Mentions:
We next measured metal levels to determine whether the copper tolerance seen with midgut PSN knockdown was caused by reduced dietary copper uptake. ICP-MS was used to measure metal levels in pupae, as this represents the end of the larval feeding stage (Figure 3, Figure S2). The weight of the PSN knockdown pupae (0.88±0.02 mg) was not significantly different to the RNAi control (0.84±0.02 mg) or GAL4 control (0.91±0.02 mg) pupae. Copper levels were measured in Drosophila reared on the copper-chelator BCS, basal media and also copper-supplemented media (Figure 3A). Zinc levels were measured in Drosophila reared on the zinc chelator TPEN, basal media and also zinc-supplemented media (Figure 3B). Relative to each control strain, midgut PSN knockdown was associated with significantly reduced copper levels when reared on copper-supplemented media and there was also a trend for lower levels on basal media (Figure 3A). Zinc levels were not consistently different between midgut PSN knockdown flies and controls when reared on zinc-limiting, basal or zinc-supplemented conditions (Figure 3B). PSN knockdown did not consistently alter basal levels of iron, manganese, calcium or magnesium (Figure S2).

Mentions:
We next measured metal levels to determine whether the copper tolerance seen with midgut PSN knockdown was caused by reduced dietary copper uptake. ICP-MS was used to measure metal levels in pupae, as this represents the end of the larval feeding stage (Figure 3, Figure S2). The weight of the PSN knockdown pupae (0.88±0.02 mg) was not significantly different to the RNAi control (0.84±0.02 mg) or GAL4 control (0.91±0.02 mg) pupae. Copper levels were measured in Drosophila reared on the copper-chelator BCS, basal media and also copper-supplemented media (Figure 3A). Zinc levels were measured in Drosophila reared on the zinc chelator TPEN, basal media and also zinc-supplemented media (Figure 3B). Relative to each control strain, midgut PSN knockdown was associated with significantly reduced copper levels when reared on copper-supplemented media and there was also a trend for lower levels on basal media (Figure 3A). Zinc levels were not consistently different between midgut PSN knockdown flies and controls when reared on zinc-limiting, basal or zinc-supplemented conditions (Figure 3B). PSN knockdown did not consistently alter basal levels of iron, manganese, calcium or magnesium (Figure S2).

Bottom Line:
Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake.SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat.These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Affiliation:
Department of Genetics, The University of Melbourne, Victoria, Australia.

ABSTRACTDietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.